kD
100
66
55
37
31
22
14
B
1 2 3 4 5 6 7 8 9
SAT activity (% of wild type)
A
100
80
60
40
20
0.2
0.1
0.0
At-SATa
At-SATi
Suppl. 1 At-SATi is more than 1000-fold less active than AtSATa
A) At-SATa and At-SATi cDNAs were cloned in the pET28a vector (Novagen, Darmstadt) for
expression of respective At-SAT protein in fusion with an N-terminal His-tag. After coexpression of At-OAS-TL C and At-SATa in Eschericia coli crude proteins were isolated in
extraction buffer (10 mM Tris pH 8, 0.25 M sodium chloride, 1 mM PMSF). The recombinant
CSC consisting of At-OAS-TLC and At-SATa was purified by immobilized metal affinity
chromatography using a His-Trap column as described by the manufacturer (GEHealthcare, Freiburg). The At-OAS-TL C protein contains no purification tag but was copurified with At-SATa upon its high affinity to At-SATa. The received fractions of this
purification were separated by SDS-gel electrophoresis (1: crude extract, 2: flow through, 3:
wash step with extraction buffer containing 80 mM imidazole, 4: elution of CSC with
extraction buffer containing 400 mM imidazole). In addition the His-trap bound CSC was
dissociated using 10 mM OAS prior to elution with imidazole (6: At-OAS-TL C eluted with 10
mM OAS, 7: At-SATa eluted with imidazole). The same purification method was applied for
complexes of inactive At-SATi protein and At-OAS-TL C (5), as expected no obvious
differences in complex stoichiometry were noticeable between At-SATa/OAS-TL C and AtSATi/OAS-TL C (comparison of 4 and 5). The dissociation of At-SATi/At-OAS-TL C
complexes could be demonstrated by treatment with OAS (8: At-OAS-TL C eluted with 10
mM OAS, 9: At-SATi eluted with imidazole).
B) Purified At-SATa (7) and At-SATi (9) were used to determine the specific SAT activity by
an uncoupled online assay system. The activity measurements rely on the absorbance of
the thioester bound in acetyl-coenzyme A at 254 nm, which is cleaved during transfer of the
acetyl-moiety from acetyl-coenzyme A to serine. The assay was performed at 25 °C for 20
min in quartz-cells (0.25 ml) and contains 0.1 M Hepes pH 7.5, 1 mM DTT, 0.2 mM acetylcoenzyme A, 5 mM serine and varying amounts of AtSAT (0.1 - 8 µg, n = 8). As expected
the specific SAT activity of At-SATa was more than 1000-fold higher than the activity of the
mutated At-SATi protein.